Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.
Abstract: Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-μm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4',6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastincollagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease; < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease; < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy. Impact statement This work demonstrated that second harmonic generation microscopy in conjunction with elastin immunofluorescence provided a comprehensive assessment of multiscale structural re-organization in healing tendon than when restricted to longitudinal collagen fiber alignment alone. Utilizing this approach for tendon histomorphometry is ideal not only to improve our understanding of hierarchical structural changes that occur after tendon injury and during remodeling but also to monitor the efficacy of therapeutic approaches.
Publication Date: 2020-05-05 PubMed ID: 32228165DOI: 10.1089/ten.TEC.2020.0032Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research focuses on a new method to analyze the healing of tendons by examining not only collagen alignment, but also the structure of non-collagenous proteins. Using second harmonic generation (SHG) imaging and elastin immunofluorescence, the study showed significant structural changes in healing tendons and suggested the need to incorporate these factors into histological evaluations.
Research Objectives
- The main aim of the study was to use SHG imaging to measure the cross-sectional architecture of the tendon fascicle. This method can provide an objective assessment of how the tendon is arranged and its changes during healing.
- The researchers also wanted to combine the SHG with elastin immunofluorescence, as elastin is another important protein in tendons. This combination could help understand the ultrastructure of collagen and elastin in the tendons.
- Lastly, they aimed to evaluate the changes in the collagen alignment of fascicles and elastin in the interfascicular matrix (IFM) in both normal and collagenase-injured tendons. This could reveal the changes that occur during healing.
Research Methodology
- The researchers collected longitudinal and cross-sectional sections from normal and collagenase-injured equine tendons, and prepared them for SHG microscopy and elastin immunofluorescence.
- They applied anti-elastin antibodies to highlight elastin structures and used a secondary antibody for visualization.
- SHG imaging and standard confocal imaging were used to visualize the collagen and elastin structures in the tendons, respectively.
- The measurements of fascicle cross-sectional area, IFM thickness, the percentage of elastin in IFM, and the alignment of elastin and collagen structures were conducted using ImageJ software.
Results and Findings
- They found significant differences in the measurements between normal and collagenase-injured tendons at 6 and 16 weeks after injury. These differences show structural changes that occur during tendon healing.
- Furthermore, the researchers found that the collagen re-alignment was happening faster than that of elastin.
- These findings highlight the importance of including IFM and non-collagenous proteins in tendon histological evaluations.
Implications of the Research
- This research suggests that SHG microscopy combined with elastin immunofluorescence can give a much comprehensive assessment of tendon healing.
- Considering both collagen and non-collagenous proteins could improve our understanding of the changes that occur after tendon injury and during remodeling.
- This approach could also be used to monitor the efficiency of therapeutic interventions for tendon injuries.
Cite This Article
APA
Durgam S, Singh B, Cole SL, Brokken MT, Stewart M.
(2020).
Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.
Tissue Eng Part C Methods, 26(5), 253-262.
https://doi.org/10.1089/ten.TEC.2020.0032 Publication
Researcher Affiliations
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
- Campus Microscopy Imaging Facility, The Ohio State University, Columbus, Ohio, USA.
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
- Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, Illinois, USA.
MeSH Terms
- Animals
- Collagenases / metabolism
- Elastin / analysis
- Elastin / metabolism
- Extracellular Matrix / chemistry
- Horses
- Microscopy, Fluorescence / methods
- Second Harmonic Generation Microscopy / methods
- Tendons / chemistry
- Tendons / pathology
Citations
This article has been cited 11 times.- Jana A, Sarkar A, Zhang H, Agashe A, Wang J, Paul R, Gov NS, DeLuca JG, Nain AS. Mitotic outcomes and errors in fibrous environments. Proc Natl Acad Sci U S A 2023 Mar 7;120(10):e2120536120.
- Jaschke M, Kolodziej L, Huebotter C, Hennecke T, Koppe D, Wilk A. An overview in tendon's physiology, pathomorphology, and treatment options. Cell Tissue Res 2026 Feb 10;403(2):17.
- Hebner TS, Genc DE, Benoit DSW. Recapitulating the Native Tendon Environment in a Synthetic 3D Anisotropic Hydrogel as an Engineered Extracellular Matrix. ACS Appl Bio Mater 2026 Feb 16;9(4):2282-2293.
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